1. Field of the Invention
This invention relates to the spike protein of infectious bronchitis virus (IBV) and to a recombinant DNA method for preparing it. IBV is a virus which causes respiratory disease in the fowl, and is of particular importance in relation to poultry.
2. Description of the Prior Art
IBV is a virus of the type Coronaviridae. It has a single-stranded RNA genome, approximately 20 kb in length, of positive polarity, which specifies the production of three major structural proteins: nucleocapsid protein, membrane glycoprotein, and spike glycoprotein. The spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus. The spike protein is believed likely to be responsible for immunogenicity of the virus, partly by analogy with the spike proteins of other corona-viruses and partly by in vitro neutralisation experiments, see, for example, D. Cavanagh et al., Avian Pathology 13, 573-583 (1984). Although the term "spike protein" is used to refer to the glycoproteinaceous material of the spike, it has recently been characterised by D. Cavanagh, Journal of General Virology 64, 1187-1191; 1787-1791; and 2577-2583 (1983) as comprising two or three copies each of two glycopolypeptides, S1 (90,000 daltons) and S2 (84,000 daltons). The polypeptide components of the glycopolypeptides S1 and S2 have been estimated after enzymatic removal of oligosaccharides to have a combined molecular weight of approximately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the S2 polypeptide.
The genomic organisation of the IBV viral proteins is summarised in, for example, T. D. K. Brown and M. E. G. Boursnell, Virus Research 1, 15-24 (1984). Briefly, six polyadenlyated IBV viral mRNA species (A to F) have been detected in infected cells. mRNA A is the smallest and mRNA F is of genome length. These mRNAs form a so-called `nested` or 3' co-terminal set. The nested mRNAs A to E have sizes approximately 2, 2.4, 3.4, 4.1 and 7.8 kb, as estimated from formaldehyde-agarose gel electrophoresis. They are shown in the accompanying drawing. Evidence from translation studies in vitro suggests that mRNAs A, C and E are each translated to give a corresponding major polypeptide. Thus, mRNA A codes for the nucleocapsid polypeptide, mRNA C for the membrane polypeptide and mRNA E for the precursor of the spike protein. In connection with mRNA E D. F. Stern and B. M. Sefton, Journal of Virology 50, 22-29 (1984) found that this mRNA specified production of the spike protein precursor in an in vitro translation. The sizes of the translation products are consistent with the coding capacity being present at the 5' end of each mRNA, but not present in the next smallest mRNA. In other words, the coding portion is within the "unique" region, i.e. the region of `non-overlap` between successive RNAs of the set. U.v. inactivation studies have demonstrated that the subgenomic mRNAs are not produced by processing of larger RNA species, but are synthesised independently.
DNA complementary to IBV RNA (hereinafter referred to as `cDNA`) has been obtained for the Beaudette strain of IBV, as two fragments, together encompassing the first 3.3 kb of RNA from the 3' end, extending nearly to the 5' end of mRNA C. The fragments were inserted in plasmids and cloned in E. coli. They are described as C5.136 and C5.322 in T. D. K. Brown and M. E. G. Boursnell, supra, C5.136 being that running from nucleotides 1000 to 3300 approximately. Sequence information on C5.136 from nucleotides 1630 to 2400 approximately and the cloning of cDNA for IBV Beaudette strain including mRNA B and the 5' region of mRNA A have been described by M. E. G. Boursnell and T. D. K. Brown, Gene 29, 87-92 (1984). Futher C5.136 sequence from nucleotides 2200 to 3400 approximately has been published by M. E. G. Boursnell, T. D. K. Brown and M. M. Binns, Virus Research 1, 303-313 (1984).
In the paper `Genetically Engineered Vaccine against Avian Infectious Bronchitis Virus with the Advantages of Current Live and Killed VAccines`, by D. Cavanagh and the present inventors (M. M. Binns, M. E. G. Boursnell and T. D. K. Brown) in `Modern Approaches to Vaccines`, Cold Spring Harbor Laboratory, New York 1984, pages 215-218, it was announced that an oligonucleotide primer had been made and was currently being used to extend the C5.136 DNA so as to encompass the spike protein precursor gene. The oligonucleotide primer was described as corresponding to a sequence of 13 nucleotides approximately 150 bases in from the 5' terminus of C5.136. The nature and exact location of the oligonucleotide in the C5.136 cDNA sequence in the region from nucleotides 2400 to 3300 (the 5' terminus) have not been disclosed by these workers in any way, in writing or orally.